This invention relates to an assay procedure and kit for use in determining the levels of FK 506 or other macrophilin binding substances in blood, particularly un-extracted blood in the presence of specific binding proteins for FK 506. The method uses derivatives of FK 506 to displace the FK 506 from specific binding proteins for FK 506. The invention also relates to novel compounds that are useful for displacing FK 506 from its specific binding protein.
FK 506 (or tacrolimus) is a cyclic, poly-N-methylated undecapeptide, possessing immunosuppressive activity. FK 506 is a macrolide immunosuppressant that is produced by Streptomyces tsukubaensis No 9993. The structure of FK 506 is shown in FIG. 1.

Also a large number of related compounds which retain the basic structure and immunological properties of FK 506 are also known. These compounds are described in a number of publications, for example EP 184162, EP 315973, EP 323042, EP 423714, EP 427680, EP 465426, EP 474126, WO 91/13889, WO 91/19495, EP 484936, EP 532088, WO 93/5059 and the like.
FK 506 compounds are used, for example, in the prevention of transplant rejection. However these FK 506 compounds have side effects at higher doses and therefore their concentration in the blood must be kept within certain therapeutic ranges. Further, bioavailabilities and metabolic conversion rates tend to be patient specific and hence dosing is patient specific. The potency and the spectrum of toxicity of FK 506 require sensitive, reproducible and reliable methods for monitoring the blood concentration of these compounds. It is therefore necessary to monitor the concentration of these immunosuppressant drugs in the blood at regular intervals. Rapamycin and cyclosporin are other compounds used in the prevention of transplant rejection. These drugs may be used singly or in combination.
It has been found that a portion of the FK 506 compound present in blood exists in the form of a FK 506 immunophilin complex. Immunophilins are a family of intracellular binding proteins that bind cyclosporins, rapamycins or FK 506 compounds. Two distinct families of immunophilins are presently known; cyclophilins which bind to cyclosporins and macrophilins which bind to rapamycins (sirolimus) and its derivatives such as everolimus and FK 506 compounds. The structures of certain immunophilins are described in Walkinshaw et al; 1992; Transplantation Proceedings, 24, 4(2), 8–13. The macrophilins which bind to FK 506 are called FK 506 binding proteins. Five members, thus far, have been reported: FKBP-12, FKBP-12.6, FKB-13, FKBP-25 and FKBP-52.
Given the importance of cyclosporins, rapamycins and FK 506 compounds as pharmaceuticals, there is a need for simple, sensitive assays to determine their concentrations in blood.
For FK 506, specific monoclonal antibodies have been developed and assay procedures based on the antibodies provided. WO 95/07468, U.S. Pat. No. 5,532,137. However, these assay procedures available require the blood or plasma sample first be extracted with a solvent (such as methanol) which is then removed by evaporation or dilution to free the FK 506 from its binding protein. The antibody is then added to the sample and the FK 506:Ab complex is detected. The assay procedure based on the specific monoclonal antibody works well but the need for the extraction step and the subsequent removal of the solvent can result in the assay becoming less sensitive and less precise if care is not taken. Therefore the assay must be carried out by skilled technicians and is a time consuming procedure.
It is known from studies conducted in vitro, excess concentrations of rapamycin prevent the effects of FK 506 by displacing FK 506 from FKBP's. Fruman, David et al., Calcineurin Phosphatase activity in T lymphocytes is inhibited bv FK 506 and cyclosporin A, PNAS (1992) 89(9), 3683–90. Further, EP 717850 discloses an assay method for FK 506 that uses a binding-competitor, namely rapamycin, to displace FK 506 from its binding protein. Thus, an extraction step is not needed. However, since rapamycin, FK 506 and cyclosporin can be used singly or in combination, it is preferred that the binding-competitor be a different compound than one of the immunosuppressive drugs so that the amount of binding competitor in the sample can be controlled.